Plant preservative mixture

Arabidopsis thaliana ecotype Col-0 constitutively expressing intracellular Ca²⁺ indicator aequorin (pMAQ2) is a gift from M. Knight and the principles of how the active aequorin is formed can be found in Knight et al. (1991) (link). Arabidopsis plants were grown in 150 mm × 15 mm round Petri dishes in half-strength Murashige and Skoog salts (MS; Gibco), supplemented with 1.5% (w/v) sucrose (Sigma), and 0.8% (w/v) agar (Becton Dickinson) adjusted to pH 6.0 with KOH in controlled an environmental room at 21 ± 2°C. The fluency rate of white light was ∼110 μmol m^-2 s^-1. The photoperiods were 16 h light/8 h dark cycles. Seeds were sterilized with 2.5% plant preservative mixture (Caisson Laboratories) and stratified at 4°C for 3 days in the dark, and then transferred to the growth room.

Catharanthus roseus seeds (Little Bright Eye) were surface‐sterilized in 70% (v/v) ethanol for one minute, followed by 10% (v/v) bleach containing 0.1% (v/v) Triton X‐100 for ten minutes. The seeds were rinsed three times in sterile water and soaked in 1% Plant Preservative Mixture (Caisson Laboratories) in sterile water for 24 hr. The seeds were planted in sterile Magenta GA‐7 boxes on the surface of 1/2 strength Murashige and Skoog media (2.2 g/L Murashige and Skoog basal salts with vitamins, 3% sucrose, 4 g/L Phytoagar, pH 5.7). Seedlings were grown in the dark at 25°C for one week and then transferred to a 16 hr of light (Erligpowht 45W LED Red Blue Lights) photoperiod for approximately 6 weeks before infection with Agrobacterium rhizogenes.

Six different endophytic bacterial strains were selected from contaminated node culture explants based on the color and growth speed. Bacteria were streaked on node culture medium (Table S1) supplemented with various antibiotics at mentioned concentrations, including neomycin (50 or 200 mg/l, PhytoTech Labs), rifampicin (50 mg/l, PhytoTech Labs), kanamycin (50 mg/l, PhytoTech Labs), timentin (200 mg/l, PhytoTech Labs) and cefotaxine (400 mg/l, PhytoTech Labs), and 0.2% PPM (Plant Preservative Mixture, Caisson Labs), and incubated at 28 °C for 5 days. For the anti-fungal test, after sterilization node segments were soaked overnight in sterile water (as control), or sterile water containing 0.2% bleach, 0.1% AAS (Antibiotic Antimycotic Solution, Sigma), 10 mg/l natamycin (PhytoTech Labs), 0.2% PPM, or 5 mg/l Benomyl (Methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate, Sigma). Next, treated nodal explants were cultured on node culture medium containing the same fungicide at the same concentration. Fungus contamination was counted after culturing for 2 weeks. ANOVA and LSD tests were performed in Microsoft Excel to determine the statistically significant difference among fungicide treatments.